Normal reproduction and maintenance of life in all mammalian species requires complex patterns of steroidogenic activities. A long-term goal of this laboratory is to understand at the biochemical level the mechanisms involved in regulating expression of genes encoding enzymes required for steroid hormone biosynthesis. Specifically we are focusing our attention on the steroid hydroxylases (cytochromes P450) and related enzymes in the adrenal cortex required for the conversion of cholesterol to glucocorticoids, mineralocorticoids and the adrenal androgens (precursors of sex hormones). Expression of genes encoding these enzymes is multifactorial involving developmental, tissue specific, cAMP-independent and cAMP-dependent mechanisms. In the adrenal cortex, cAMP-dependent regulation of steroid hydroxylase expression is responsive to the peptide hormone ACTH derived from the anterior pituitary, and is required for maintenance of optimal steroidogenic activity such that biosynthesis of essential steroid hormones is achieved on demand. In this competing continuation application, we propose to investigate the unique transcription factors associated with cAMP-dependent transcription of the human 45Oc2l gene, the bovine P450c17 and P45Oscc genes, and the bovine adrenodoxin gene. Previous studies in this laboratory have established that DNA-elements (CRS sequences) associated with each of these genes are unique, both with respect to one another and to other known cAMP responsive genes. We propose purification, cloning nd characterization of the transcription factors associated with each of these CRS elements. Using deletion and site-directed mutagenesis, bacterial expression of recombinant proteins, in vitro transcription systems, and transient transfection studies we will identify and characterize the DNA-binding and transcription activation domains in each of the transcription factors. To evaluate cAMP-responsive domains we will utilize (32P]-labeling, peptide mapping and protein sequencing to locate phosphoamino acids. In our long- term plan to understand at the biochemical level the chronic action of ACTH to regulate steroidogenic capacity in the adrenal cortex, these studies will elucidate sites of action of cAMP-dependent protein kinase, a key common enzyme in regulation of each of these genes.